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PubMed2001

Expression profiling using microarrays fabricated by an ink-jet oligonucleotide synthesizer.

We describe a flexible system for gene expression profiling using arrays of tens of thousands of oligonucleotides synthesized in situ by an ink-jet printing method employing standard phosphoramidite chemistry. We have characterized the dependence of hybridization specificity and sensitivity on parameters including oligonucleotide length, hybridization stringency, sequence identity, sample abundance, and sample preparation method. We find that 60-mer oligonucleotides reliably detect transcript ratios at one copy per cell in complex biological samples, and that ink-jet arrays are compatible with several different sample amplification and labeling techniques. Furthermore, results using only a single carefully selected oligonucleotide per gene correlate closely with those obtained using complementary DNA (cDNA) arrays. Most of the genes for which measurements differ are members of gene families that can only be distinguished by oligonucleotides. Because different oligonucleotide sequences can be specified for each array, we anticipate that ink-jet oligonucleotide array technology will be useful in a wide variety of DNA microarray applications.

Cells, Cultured↗

PubMed2006

Detection of small genomic imbalances using microarray-based multiplex amplifiable probe hybridization.

Array-based genome-wide screening methods were recently introduced to clinical practice in order to detect small genomic imbalances that may cause severe genetic disorders. The continuous advancement of such methods plays an extremely important role in diagnostic genetics and medical genomics. We have modified and adapted the original multiplex amplifiable probe hybridization (MAPH) to a novel microarray format providing an important new diagnostic tool for detection of small size copy-number changes in any locus of human genome. Here, we describe the new array-MAPH diagnostic method and show proof of concept through fabrication, interrogation and validation of a human chromosome X-specific array. We have developed new bioinformatic tools and methodology for designing and producing amplifiable hybridization probes (200-600 bp) for array-MAPH. We designed 558 chromosome X-specific probes with median spacing 238 kb and 107 autosomal probes, which were spotted onto microarrays. DNA samples from normal individuals and patients with known and unknown chromosome X aberrations were analyzed for validation. Array-MAPH detected exactly the same deletions and duplications in blind studies, as well as other unknown small size deletions showing its accuracy and sensitivity. All results were confirmed by fluorescence in situ hybridization and probe-specific PCR. Array-MAPH is a new microarray-based diagnostic tool for the detection of small-scale copy-number changes in complex genomes, which may be useful for genotype-phenotype correlations, identification of new genes, studying genetic variation and provision of genetic services.

Chromosome Aberrations↗

PubMed2006

Crystal structure of pi initiator protein-iteron complex of plasmid R6K: implications for initiation of plasmid DNA replication.

We have determined the crystal structure of a monomeric biologically active form of the pi initiator protein of plasmid R6K as a complex with a single copy of its cognate DNA-binding site (iteron) at 3.1-A resolution. The initiator belongs to the family of winged helix type of proteins. The structure reveals that the protein contacts the iteron DNA at two primary recognition helices, namely the C-terminal alpha4' and the N-terminal alpha4 helices, that recognize the 5' half and the 3' half of the 22-bp iteron, respectively. The base-amino acid contacts are all located in alpha4', whereas the alpha4 helix and its vicinity mainly contact the phosphate groups of the iteron. Mutational analyses show that the contacts of both recognition helices with DNA are necessary for iteron binding and replication initiation. Considerations of a large number of site-directed mutations reveal that two distinct regions, namely alpha2 and alpha5 and its vicinity, are required for DNA looping and initiator dimerization, respectively. Further analysis of mutant forms of pi revealed the possible domain that interacts with the DnaB helicase. Thus, the structure-function analysis presented illuminates aspects of initiation mechanism of R6K and its control.

Amino Acid Sequence↗

PubMed1986

Early postimplantation embryo lethality due to DNA rearrangements in a transgenic mouse strain.

Insertional mutagenesis in a transgenic mouse strain (HUGH/3) was caused by integration of plasmid DNA containing the human growth hormone gene and pBR322 plasmid sequences. From this study, which includes another instance of mutagenesis, and from other reports, it is apparent that insertional mutagenesis occurs fairly frequently during DNA integration in the mouse egg and that it is not specific for the exogenous DNA employed. The mutation in HUGH/3 is recessive and results in death of embryos homozygous for the donor sequences shortly after implantation, at the egg cylinder stage on days 4-5 of gestation. Restriction mapping of the insert and of the flanking DNA regions indicates that integration must have involved a series of complex events. Approximately five copies of plasmid sequences are arrayed in tandem but are interrupted at least twice by mouse cellular sequences. In addition, the mouse flanking DNA shows extensive rearrangements, probably including a deletion of at least 10 kilobases. The rearrangements may reflect an initially unstable DNA structure followed by attainment of a more stable conformation.

Animals↗

PubMed1990

Unusual molecular characteristics of a repeat sequence island within a Giemsa-positive band on the mouse X chromosome.

The mouse genome contains 50 copies of a long complex repeat unit localized as a repeat sequence island to the A3 Giemsa-positive (dark) band on the mouse X chromosome. The repeat units are not tandemly arranged but are juxtaposed and inserted by unrelated sequences of high repetition. The repeat sequence island possesses two notable features that have been suggested as diagnostic features of mammalian Giemsa-positive bands. First, the repeat sequence island encompasses a 1-megabase region devoid of CpG islands; second, it features a high concentration of L1 long interspersed repeat sequences.

Animals↗

PubMed1997

Nuclear punctate distribution of ALL-1 is conferred by distinct elements at the N terminus of the protein.

The ALL-1 gene positioned at 11q23 is directly involved in human acute leukemia either through a variety of chromosome translocations or by partial tandem duplications. ALL-1 is the human homologue of Drosophila trithorax which plays a critical role in maintaining proper spatial and temporal expression of the Antennapedia-bithorax homeotic genes determining the fruit fly's body pattern. Utilizing specific antibodies, we found that the ALL-1 protein distributes in cultured cells in a nuclear punctate pattern. Several chimeric ALL-1 proteins encoded by products of the chromosome translocations and expressed in transfected cells showed similar speckles. Dissection of the ALL-1 protein identified within its approximately 1,100 N-terminal residues three polypeptides directing nuclear localization and at least two main domains conferring distribution in dots. The latter spanned two short sequences conserved with TRITHORAX. Enforced nuclear expression of other domains of ALL-1, such as the PHD (zinc) fingers and the SET motif, resulted in uniform nonpunctate patterns. This indicates that positioning of the ALL-1 protein in subnuclear structures is mediated via interactions of ALL-1 N-terminal elements. We suggest that the speckles represent protein complexes which contain multiple copies of the ALL-1 protein and are positioned at ALL-1 target sites on the chromatin. Therefore, the role of the N-terminal portion of ALL-1 is to direct the protein to its target genes.

Amino Acid Sequence↗

PubMed1997

Dystrophin-glycoprotein complex is monomeric and stabilizes actin filaments in vitro through a lateral association.

The native molecular weight of the dystrophin-glycoprotein complex and its effect on actin depolymerization and polymerization were examined. First, we determined that the native molecular weight of purified dystrophin-glycoprotein complex is only large enough (Mr 1,200,000) to contain one copy of each protein in the complex, including dystrophin. Using different approaches, we also demonstrated that dystrophin-glycoprotein complex significantly protected a fraction of actin filaments from disassembly, while individual recombinant actin binding fragments of dystrophin or calpain-digested dystrophin-glycoprotein complex had no effect on F-actin depolymerization. The protective effect of dystrophin-glycoprotein complex on F-actin depolymerization saturated at a dystrophin:actin molar ratio of 0.04, corresponding to 1 dystrophin/25 actin monomers, which is highly consistent with the 1:24 stoichiometry of dystrophin-glycoprotein complex binding to F-actin previously measured at equilibrium. However, dystrophin-glycoprotein complex did not bind G-actin or alter the kinetics or extent of actin polymerization. This excluded the possibility that dystrophin-glycoprotein complex inhibited actin depolymerization by capping the ends of actin filaments. It therefore appears that actin binding domains separated on the dystrophin molecule from each other by almost 1,200 amino acids act in concert to protect F-actin from depolymerization. Our data suggest that dystrophin stabilizes F-actin in vitro by binding alongside an actin filament and bridging actin monomers in a manner analogous to the actin side binding protein tropomyosin. It is noteworthy that we did not find any effect of skeletal muscle tropomyosin on dystrophin-glycoprotein complex binding to F-actin. This indicates that dystrophin-glycoprotein complex and tropomyosin may simultaneously bind the same actin filament and identifies another feature that distinguishes dystrophin from the other proteins in the actin-cross-linking superfamily.

Actins↗

PubMed2006

Structures of the carbohydrate recognition domain of Ca2+-independent cargo receptors Emp46p and Emp47p.

Emp46p and Emp47p are type I membrane proteins, which cycle between the endoplasmic reticulum (ER) and the Golgi apparatus by vesicles coated with coat protein complexes I and II (COPI and COPII). They are considered to function as cargo receptors for exporting N-linked glycoproteins from the ER. We have determined crystal structures of the carbohydrate recognition domains (CRDs) of Emp46p and Emp47p of Saccharomyces cerevisiae, in the absence and presence of metal ions. Both proteins fold as a beta-sandwich, and resemble that of the mammalian ortholog, p58/ERGIC-53. However, the nature of metal binding is distinct from that of Ca(2+)-dependent p58/ERGIC-53. Interestingly, the CRD of Emp46p does not bind Ca(2+) ion but instead binds K(+) ion at the edge of a concave beta-sheet whose position is distinct from the corresponding site of the Ca(2+) ion in p58/ERGIC-53. Binding of K(+) ion to Emp46p appears essential for transport of a subset of glycoproteins because the Y131F mutant of Emp46p, which cannot bind K(+) ion fails to rescue the transport in disruptants of EMP46 and EMP47 genes. In contrast the CRD of Emp47p binds no metal ions at all. Furthermore, the CRD of Emp46p binds to glycoproteins carrying high mannosetype glycans and the is promoted by binding not the addition of Ca(2+) or K(+) ion in These results suggest that Emp46p can be regarded as a Ca(2+)-independent intracellular lectin at the ER exit sites.

Amino Acid Sequence↗

PubMed2003

Agraphia with abnormal writing stroke sequences due to cerebral infarction.

A 65-year-old, right-handed man presented with speech and gait disturbances. He was alert and cooperative, showing mild right hemiparesis and sensory disturbance. Spontaneous speech was fluent; object naming, word fluency and reading were fully preserved. Sentence repetition and verbal comprehension were mildly impaired. Writing was slow, hesitant and difficult for both spontaneous writing and dictation. Copying was better, although he had some difficulty in copying letters and also complex figures. Sequences of strokes in forming written characters were abnormal; strokes were formed by piecing together several fragments. Computed tomography and magnetic resonance imaging showed a subcortical infarct in the left frontoparietal region. Characteristics of agraphia resembled 'apractic agraphia' and agraphia may have resulted largely from loss or unavailability of the memory of motor patterns necessary for writing letters.

Aged↗

PubMed2006

Two mammalian Sec16 homologues have nonredundant functions in endoplasmic reticulum (ER) export and transitional ER organization.

Budding yeast Sec16 is a large peripheral endoplasmic reticulum (ER) membrane protein that functions in generating COPII transport vesicles and in clustering COPII components at transitional ER (tER) sites. Sec16 interacts with multiple COPII components. Although the COPII assembly pathway is evolutionarily conserved, Sec16 homologues have not been described in higher eukaryotes. Here, we show that mammalian cells contain two distinct Sec16 homologues: a large protein that we term Sec16L and a smaller protein that we term Sec16S. These proteins localize to tER sites, and an N-terminal region of each protein is necessary and sufficient for tER localization. The Sec16L and Sec16S genes are both expressed in every tissue examined, and both proteins are required in HeLa cells for ER export and for normal tER organization. Sec16L resembles yeast Sec16 in having a C-terminal conserved domain that interacts with the COPII coat protein Sec23, but Sec16S lacks such a C-terminal conserved domain. Immunoprecipitation data indicate that Sec16L and Sec16S are each present at multiple copies in a heteromeric complex. We infer that mammalian cells have preserved and extended the function of Sec16.

Amino Acid Sequence↗

PubMed1993

Determination of DNA replication kinetics in synchronized human cells using a PCR-based assay.

Studies on the temporal order of DNA replication are difficult due to the lack of sensitivity of methods available for replication kinetic analysis. To overcome problems associated with the current techniques, we propose a PCR-based assay to determine the replication time of any single-copy DNA sequence in complex genomes. Human cells labeled with 5-bromodeoxyuridine (BrdU) were flow sorted, according to their DNA content, at different times after synchronous release from the G1/S phase boundary. The selective removal of newly-replicated BrdU-substituted DNA was achieved by UV light irradiation followed by S1 nuclease treatment. The timing of replication of selected DNA sequences (housekeeping, tissue-specific, and non-coding loci) was determined by polymerase chain reaction (PCR) amplification using appropriate primers. DNA sequences localized in inactive replication units allowed amplification whereas those that have replicated will not be amplified by PCR. Using this sensitive and quantitative assay the replication kinetic analysis of a number of different DNA sequences can be performed from a single sorting experiment.

Bromodeoxyuridine↗

PubMed1980

Saccharomyces cerevisiae plasmid, Scp or 2 mum: intracellular distribution, stability and nucleosomal-like packaging.

Cell fractions from yeast strains known to harbor the plasmic 2 mum or Scp were treated with nucleases used to probe eukaryotic chromosome structure. Scp and subfragments were identified by hybridization to natural or cloned Scp probes according to Southern (34). Specificity was confirmed with non-Scp probes. Copy/haploid nuclear genome(n) was estimated from reconstructions at a resolution of 0.5/n. About 43-67% of the total cellular copy exists as nucleoprotein complexes which separate from other debris on isokinetic sucrose gradients with s-values of 67-110. These complexes are totally degraded by DNAase I. Digestion with micrococcal nuclease produced integral-sized fragments; they are not generated by direct mixing of pure Scp with nuclear chromatin from a[cir] strain. Initial digests gave a repeat of 168 +/- 3 base pairs (bp) for both Scp and nuclear nucleoprotein; advanced digests reduced the nuclear repeat relative to Scp by 8 bp. Of a potential 37 repeat units/plasmid, 31-32 were directly measured. A strain difference in Scp autodegradation was found. A partial nuclease resistant form was also demonstrated whose abundance was cell strain and growth stage dependent. Both Scp isomers exist in these complexes which are structurally similar to simian viral 40 minichromosomes.

Deoxyribonuclease I↗

PubMed2026

DNA copy number patterns reveal prognostic markers and elucidate mechanisms of evolution in IDH-mutant astrocytoma.

BACKGROUND: Current literature suggestsisocitrate dehydrogenase (IDH)-mutant astrocytoma contains several molecular subgroups. In this study, we are interested in determining the connection between different molecular subgroups with grade and/or survival. METHODS: A cohort of 470 Mayo Clinic adult patients (≥18 years, 56.2% male) with primary IDH-mutant astrocytoma diagnosed by World Health Organization (WHO) 2021 criteria were examined. Results were validated in an independent cohort of 614 Mayo Clinic Neuropathology consult patients and 235 The Cancer Genome Atlas (TCGA) patients. RESULTS: The Mayo Clinic Practice cohort confirmed the association of CDKN2A/B deletion with overall survival (OS, homozygous vs hemizygous vs intact, 2.7 vs 9.6 vs 17.2 years, P < .001). Phosphatase and tensin homolog (PTEN) deletion was also associated with poor OS (7.3 vs 17.4 years, P < .001). Increased number of copy number alterations was associated with OS (continuous variable, HR = 1.027, P < .001). Carrying one or more copies of the germline risk allele at rs55705857 was associated with earlier age of onset (median age 33 vs 35 years, P = .01), and a shorter OS after adjusting for age, grade, sex and treatment (HR = 1.81, P = .007). The Mayo Clinic Neuropathology Consult cohort and TCGA were utilized to validate age of onset and survival, respectively. Unsupervised clustering of the copy number alterations identified several clinically significant groups that may define pathways to disease progression. Losses of chromosomes 11p, 13q, 1p, and 10q were all associated with reduced overall survival in the Mayo Clinic cohort. CONCLUSIONS: Patients with hemizygous loss of CDKN2A/B, loss of PTEN, increased number of copy number alterations, specific chromosomal arm losses or rs55705857 germline risk allele have reduced overall survival.

Humans↗

PubMed1980

Ribosomal RNA genes in Kluyveromyces marxianus.

DNA from the yeast Kluyveromyces marxianus was studied for its heterogeneity and the multiplicity of rRNA cistrons. These cistrons banded at a slightly different density from the bulk DNA in preparative CsCl or Hg2+-Cs2SO4 equilibrium density gradients. The reassociation kinetics of the bulk DNA showed that the repetitive fraction represented a small amount of the total cellular DNA (10%) and that the single copy fraction had a complexity of 6.3 X 10(9) daltons. Approximately 2.2% of the DNA hybridized to 3H-labelled rRNA at saturation. On the basis of the above genome size, the multiplicity of the rRNA cistrons was calculated to be about 70 per haploid nucleus.

Base Sequence↗

PubMedDate not supplied

Middle-school-age outcomes in children with very low birthweight.

Most previous studies of children with birthweight <750 g have focused on early childhood sequelae. To evaluate later outcomes, a regional sample of 60 <750-g birthweight children was compared at middle school age (M = 11 years) to 55 children with birthweight 750-1,499 g and 49 term controls. The groups were matched on age, gender, and demographic variables at the time of an early-school-age assessment (mean age 7 years). The <750-g birthweight group fared less well at middle school age than the term group on measures of cognitive function, achievement, behavior, and academic performance. In many instances, outcomes were less favorable for the <750-g children than for the 750 to 1,499-g group. Children in the <750-g group who were free of neurosensory disorders and global cognitive impairment performed more poorly on several tests than their term counterparts. Group differences in this subsample on tests of motor skills, math, and the ability to copy and recall a complex drawing remained significant even after controlling for IQ. Disparities between the <750-g and term groups increased with age for some measures. Despite favorable outcomes for many children in the <750-g group, this population is at risk for long-term developmental problems.

Attention↗

PubMed2000

Molecular aspects of the cellular activities of ADP-ribosylation factors.

Adenosine diphosphate-ribosylation factor (Arf) proteins are members of the Arf arm of the Ras superfamily of guanosine triphosphate (GTP)-binding proteins. Arfs are named for their activity as cofactors for cholera toxin-catalyzed adenosine diphosphate-ribosylation of the heterotrimeric G protein Gs. Physiologically, Arfs regulate membrane traffic and the actin cytoskeleton. Arfs function both constitutively within the secretory pathway and as targets of signal transduction in the cell periphery. In each case, the controlled binding and hydrolysis of GTP is critical to Arf function. The activities of some guanine nucleotide exchange factors (GEFs) and guanosine triphosphatase (GTPase)-activating proteins (GAPs) are stimulated by phosphoinositides, including phosphatidylinositol 3,4,5-trisphosphate (PIP3) and phosphatidylinositol 4,5-bisphosphate (PIP2), and phosphatidic acid (PA), likely providing both a means to respond to regulatory signals and a mechanism to coordinate GTP binding and hydrolysis. Arfs affect membrane traffic in part by recruiting coat proteins, including COPI and clathrin adaptor complexes, to membranes. However, Arf function likely involves many additional biochemical activities. Arf activates phospholipase D and phosphatidylinositol 4-phosphate 5-kinase with the consequent production of PA and PIP2, respectively. In addition to mediating Arf's effects on membrane traffic and the actin cytoskeleton, PA and PIP2 are involved in the regulation of Arf. Arf also works with Rho family proteins to affect the actin cytoskeleton. Several Arf-binding proteins suspected to be effectors have been identified in two-hybrid screens. Arf-dependent biochemical activities, actin cytoskeleton changes, and membrane trafficking may be integrally related. Understanding Arf's role in complex cellular functions such as protein secretion or cell movement will involve a description of the temporal and spatial coordination of these multiple Arf-dependent events.

ADP-Ribosylation Factors↗

PubMed1975

Binding of bacteriocin Clo DF13 to Clo DF13 plasmid deoxyribonucleic acid in vivo and in vitro.

The bacteriocinogenic plasmid Clo DF13 is present in Escherichia coli to the extent of 10 copies per cell. A complex of Clo DF13 plasmid deoxyribonucleic acid (DNA) and protein can be isolated from cells. Treatment of the complex with ionic detergents or proteases dissociates the complex but does not convert any supercoiled Clo DF13 DNA to the open circular form, indicating that this complex is not a relaxation complex. The complex is stable in 0.5 M NaCl and contains one polypeptide species. The protein, present in the complex, appeared to be bacteriocin Clo DF13 for the following reasons: (i) the protein is de novo synthesized in Clo DF13-harboring minicells, indicating that this protein is Clo DF13 specific; (ii) this protein shows bacteriocinogenic activity on a bacteriocin Clo DF13-susceptible indicator strain; (iii) this protein has the same molecular weight (60,000) as bacteriocin Clo DF13. DNA-protein binding experiments, involving QAE-Sephadex column chromatography and nitrocellulose membrane filters, demonstrate that bacteriocin Clo DF13 has also affinity in vitro for Clo DF13 DNA. Membrane filter binding experiments revealed that bacteriocin Clo DF13 does not interact with other DNA species, such as ColE1 DNA, yeast DNA, calf thymus DNA, phiX174 DNA, and also not with denatured Clo DF13 DNA. In addition no binding to Clo DF13 DNA of a related bacteriocin, colicin E3, could be detected. These results indicate that the binding of bacteriocin Clo DF 13 to double-stranded Clo DF13 DNA is very specific.

Bacterial Proteins↗

PubMed2004

Identification and molecular characterization of a gene encoding a protective Leishmania amazonensis Trp-Asp (WD) protein.

Several Leishmania proteins have been identified and characterized in pursuit of understanding pathogenesis and protection in cutaneous leishmaniasis. In the present study, we utilized sera from infected BALB/c mice to screen a Leishmania amazonensis amastigote cDNA expression library and obtained the full-length gene that encodes a novel Trp-Asp (WD) protein designated LAWD (for Leishmania antigenic WD protein). The WD family of proteins mediates protein-protein interactions and coordinates the formation of protein complexes. The single-copy LAWD gene is transcribed as a approximately 3.1-kb mRNA in both promastigotes and amastigotes, with homologues being detected in several other Leishmania species. Immunoelectron microscopy revealed a predominant localization of the LAWD protein in the flagellar pocket. Analyses of sera from human patients with cutaneous and mucocutaneous leishmaniasis indicated that these individuals mounted significant humoral responses against LAWD. Given that recombinant LAWD protein elicited the production of high levels of gamma interferon, but no detectable levels of interleukin-10 (IL-10), in CD4(+) cells of L. amazonensis-infected mice, we further examined whether LAWD could elicit protective immunity. DNA vaccination with the LAWD and IL-12 genes significantly delayed lesion development, which correlated with a dramatic reduction in parasite burdens. Thus, we have successfully identified a promising vaccine candidate and antigenic vehicle to aid in the dissection of the complicated pathogenic immune response of L. amazonensis.

Amino Acid Sequence↗